Ageing is associated with diminished apoptotic cell clearance in vivo

In this study, normal adult mice carried B220(high) conventional B cells in the spleen and liver, but carried both B220(high) and B220(low) in the bone marrow. However, at the neonatal stage, only B220(low) unconventional B cells were found in all these organs. This pattern continued up to 2 weeks after birth, and at this stage autoantibodies were detected in the sera. This phenomenon was seen in all tested young mice (1-2 weeks), irrespective of their gender. Furthermore, at older stages (more than 20 weeks), B220(low) cells reappeared in the spleen and liver, and these B220(low) cells became dominant in the bone marrow. Autoantibodies also reappeared in the sera of these older mice. Cell-sorting experiments revealed that B220(low) cells were able to produce autoantibodies upon lipopolysaccharide stimuli in vitro. These results suggest that B220(low) cells appear at both neonatal and older stages as physiological responses and eventually produce autoantibodies.     

Tissue-specific expansion of NKT and CD5+B cells at the onset of autoimmune disease in (NZBxNZW)F1 mice

Natural killer T (NKT) cells and CD5(+)B cells were searched for in various immune organs of autoimmune prone (NZBxNZW)F(1) (NZB/W F(1)) mice. The number of lymphocytes increased in the liver, spleen, and peritoneal cavity after the onset of disease (at the age of 30 weeks) while the number of thymocytes decreased at that time. Prominent changes of lymphocyte subsets were seen in the liver and peritoneal cavity, namely, expansion of IL-2Rbeta(+)TCRalpha beta(int) cells in the liver and of CD5(+)B220(+) cells in the peritoneal cavity. The majority of TCRalpha beta(int) cells in the liver were NK1.1(+), and CD5(+)B cells in the peritoneal cavity were CD1d(+). Proteinuria became prominent in NZB/W F(1) mice with the progression of disease. In parallel with this progression, the proportion of NKT cells decreased slightly in the liver, but their absolute number remained at a high level in this organ. These NKT cells were CD4(+) and used an invariant chain of Valpha14Jalpha281 for TCRalpha. Reflecting the elevation of CD5(+)B cells, autoantibodies against hepatocyte cytoplasmand denatured DNA were detected in sera. Although NKT cells are known to be immunoregulatory cells in some autoimmune mice, the present results raise the possibility that NKT cells as well as CD5(+)B cells might be associated with the onset of autoimmune diseases in NZB/W F(1) mice. Indeed, NKT cells in F(1) mice had a high potential to induce autoimmune-like inflammationwhen alpha-galactosylceramide was administered or when active NKT cells were transferred into young F(1) mice.     

Coincidence of autoantibody production with the activation of natural killer T cells in α-galactosylceramide-mediated hepatic injury

Natural killer T (NKT) cells are known to be specifically activated by α-galactosylceramide (α-GalCer) via their interaction with CD1d. At that time, NKT cells mediate autoreactivity and eventually induce hepatic injury. As these immune responses resemble acute autoimmune hepatitis, it was examined whether autoantibody production and the activation of autoantibody-producing B-1 cells were accompanied by this phenomenon. Autoantibodies against Hep-2 cells and double-stranded DNA were detected in sera as early as day 3 (showing a peak at day 14) when mice were treated with α-GalCer. On day 3, B220(low) cells appeared in the liver. These B220(low) cells were CD5(-) (i.e. B-1b cells) and CD69(+) (an activation marker). Primarily, such B220(low) cells were present in the peritoneal cavity, but the proportion of B220(low) cells increased with the administration of α-GalCer even at this site. In parallel with the appearance of B220(low) cells in the liver, hepatic lymphocytes acquired the potential to produce autoantibodies in in vitro cell culture in the presence of lipopolysaccharide. These results suggested that hepatic injury induced by α-GalCer administration resembled acute autoimmune hepatitis and that the major effector lymphocytes were NKT cells with autoreactivity and autoantibody-producing B-1 cells.     

NKT cells play critical roles in the induction of oral tolerance by inducing regulatory T cells producing IL-10 and transforming growth factor beta, and by clonally deleting antigen-specific T cells

Oral tolerance is the systemic unresponsiveness induced by orally administered proteins. To explore the roles of natural killer T (NKT) cells in oral tolerance, we induced oral tolerance to ovalbumin (OVA) in NKT cell-deficient mice. In CD1d-/- mice, the induction of tolerance to orally administered high- or low-dose OVA was impaired. Dendritic cells (DCs) in the Peyer's patches (PPs) of CD1d-/- mice fed OVA showed high expression of major histocompatibility complex (MHC) class II and B7 molecules, whereas DCs of control mice fed OVA expressed low levels of these molecules. The adoptive transfer of NKT cells restored oral tolerance and induction of tolerogenic DCs in the PPs and spleens of CD1d-/- mice. Moreover, interleukin (IL)-10 and transforming growth factor (TGF)-beta1 production in vitro were reduced in cells from the spleen and PPs of CD1d-/- mice compared with those of control mice fed OVA. The numbers of OVA-specific CD4+ KJ1-26+ T cells were significantly reduced in the PPs and spleens of DO11.10 mice fed OVA. In contrast, OVA-specific CD4+ KJ1-26+ T cells were not deleted in the PPs or spleens of DO11.10 CD1d-/- mice. In conclusion, NKT cells were found to play an indispensable role in oral tolerance by inducing regulatory T cells, and clonally deleting antigen-specific CD4+ T cells.     

Simultaneous activation of natural killer T cells and autoantibody production in mice injected with denatured syngeneic liver tissue

Denatured syngeneic liver tissue prepared by mechanical procedures was intraperitoneally injected into adult C57BL/6 mice. In parallel with a decrease in the total number of lymphocytes in the liver, spleen, and thymus from days 1-7 after the injection, the proportion of the CD4+NK1.1+CD3(int) subset of these cells (i.e. natural killer T or NKT cells) increased in the liver. Even the absolute number of these NKT cells increased in the liver on days 14 and 21. In response to the injection of denatured liver tissue, tissue damage was induced in the liver, as shown by elevated levels of serum transaminases and hepatocyte degeneration observed by electron microscopy. Sera obtained on days 7 and 14 contained autoantibodies including anti-DNA antibodies. The proportion of CD1d(high)B cells in the liver was found to decrease on days 1-7. In other words, denatured liver tissue stimulated both NKT cells and certain B cells in the liver. These results suggest that liver lymphocytes might contain not only autoreactive T cells (e.g. CD3(int) or NKT cells) but also some B cells (e.g. B-1 cells) which produce autoantibodies and that the denatured tissue had the potential to stimulate these lymphocytes and to evoke an autoimmune-like state.     

Age-dependent variation in the proportion and number of intestinal lymphocyte subsets, especially natural killer T cells, double-positive CD4+ CD8+ cells and B220+ T cells, in mice

The age-dependent variation in the proportion and number of lymphocyte subsets was examined at various extrathymic sites, including the liver, small intestine, colon and appendix in mice. In comparison with young mice (4 weeks of age), the number of total lymphocytes yielded by all tested organs was greater in adult (9 weeks) and old (40 weeks) mice. The major lymphocyte subset that expanded with age was interleukin-2 receptor (IL-2R) beta+ CD3int cells (50% of them expressed NK1.1) in the liver, whereas it was CD3+ IL-2Rbeta- NK1.1- cells at all intraepithelial sites in the intestine. Although NK1.1+ CD3+ cells were present at intraepithelial sites in the intestine, the proportion of this subset was rather low. The ratio of CD4 to CD8 tended to decrease among natural killer T (NKT) cells and T cells at all intraepithelial sites in the intestine with age. A unique population of double-positive CD4+ CD8+ cells in the small intestine increased in old mice. B220+ T cells were found mainly in the appendix and colon, and the proportion of these T cells decreased in old mice. Conventional NKT cells were very few in Jalpha281-/- and CD1d-/- mice in the liver, while NKT cells which existed in the appendix remained unchanged even in these mice. This was because unconventional CD8+ NKT cells were present in the intestine. The present results suggest that despite the fact that both the liver and intraepithelial sites in the intestine carry many extrathymic T cells, the distribution of lymphocyte subsets and their age-associated variation are site-specific.     

Induction of experimental autoimmune thyroiditis in B cell-depleted mice

The effect of B cell depletion on the induction and severity of murine experimental autoimmune thyroiditis was investigated. Thirteen CBA mice were given repeated intraperitoneal doses of 700 micrograms purified rabbit anti-mouse Ig antibody from 24 hours to 8 weeks after birth. Controls were given normal rabbit IgG (14 mice) or were left uninjected (10 mice). At six weeks all mice received two doses of 70 micrograms murine thyroid extract in complete Freund's adjuvant. Only 2/13 of the anti-Ig treated mice were fully B cell-deficient as determined by serum IgM, spleen cell immunofluorescence and responsiveness to LPS; however, the levels of anti-thyroglobulin autoantibodies were very low in 7/13 mice. The results demonstrate that thyroiditis can be actively induced in the absence of B cells and autoantibodies but that B cells may play a role in increasing disease severity.     

B Cells are Selectively Associated with Thymic Cortical but not Medullary Pathology in NZB Mice

Abnormal expansion of autoantibody-synthesizing B cells and self-reactive T cells, which most likely escape negative selection within the thymus, have both been characterized and reasoned to play a role in the pathogenesis of autoimmunity in NZB mice. Support for this thesis has been our observation that NZB mice have severe cortical and medullary thymic microarchitectural defects. As a means to dissect the roles of T and B cells in the induction of such abnormalities, B cell-deficient NZB mice were bred by backcrossing the Igh6(null)allele on to the NZB background (NZB-muMT mice). Such mice showed undetectable levels of autoantibodies. NZB-muMT mice, as compared to wild-type NZB mice, had lower absolute numbers of CD4(+)T cells. Furthermore, thymic abnormalities in NZB-muMT mice were restricted to the medulla. These data suggest that, while B cells may play a role in thymic cortical abnormalities, the medullary abnormalities are induced by other mechanisms.     

Hepatic B cells are readily activated by Toll‐like receptor‐4 ligation and secrete less interleukin‐10 than lymphoid tissue B cells

B cells perform various immunological functions that include production of antibody, presentation of antigens, secretion of multiple cytokines and regulation of immune responses mainly via their secretion of interleukin (IL)‐10. While the liver is regarded both as an important immune organ and a tolerogenic environment, little is known about the functional biology of hepatic B cells. In this study we demonstrate that, following lipopolysaccharide (LPS) stimulation in vivo, normal mouse hepatic B cells rapidly increase their surface expression of CD39, CD40, CD80 and CD86, and produce significantly elevated levels of proinflammatory interferon (IFN)‐γ, IL‐6 and tumour necrosis factor (TNF)‐α compared with splenic B cells. Moreover, LPS‐activated hepatic B cells produce very low levels of IL‐10 compared with activated splenic B cells that produce comparatively high levels of this immunosuppressive cytokine. Splenic, but not hepatic, B cells inhibited the activation of liver conventional myeloid dendritic cells (mDCs). Furthermore, compared with the spleen, the liver exhibited significantly smaller proportions of B1a and marginal zone‐like B cells, which have been shown to produce IL‐10 upon LPS stimulation. These data suggest that, unlike in the spleen, IL‐10‐producing regulatory B cells in the liver are not a prominent cell type. Consistent with this, when compared with liver conventional mDCs from B cell‐deficient mice, those from B cell‐competent wild‐type mice displayed enhanced expression of the cell surface co‐stimulatory molecule CD86, greater production of proinflammatory cytokines (IFN‐γ, IL‐6, IL‐12p40) and reduced secretion of IL‐10. These findings suggest that hepatic B cells have the potential to initiate rather than regulate inflammatory responses.     

Expression and localization of fibroblast growth factor (FGF)23 and Klotho in the spleen: its physiological and functional implications

The FGF23–Klotho signaling axis is known to exert anti-aging effects via calcium–phosphorus metabolism. In mice deficient in FGF23–Klotho signaling, however, the number of splenocytes is reduced. FGF23 is expressed in both bone and spleen, with regulation of its production differing in these organs. As FGF23–Klotho signaling may play an immunological role in the spleen, splenocytes in male C57BL/6J mice were assayed for expression of Klotho or FGF23 by flow cytometry and immunohistochemistry. Cells that expressed Klotho included CD45R/B220⁺ CD21/CD35⁺ CD1d⁺ CD43^? marginal zone B cells. These cells also expressed FGF receptor 1, indicating that Klotho-positive B cells could respond to FGF23. Plasmacytoid dendritic cells (pDCs) with CD11c⁺ CD45R/B220⁺ CD11b^? CD8α^? were found to produce FGF23. Klotho-positive cells and FGF23-producing cells were present in close proximity to each other, suggesting that FGF23 produced by pDCs may act within a limited area. These findings indicate that FGF23–Klotho signaling could play a biological or immunological role in the spleen.     

The significance of T cells, B cells, antibodies and macrophages against encephalomyocarditis (EMC)-D virus-induced diabetes in mice

In order to clarify the significance of protective mechanisms against encephalomyocarditis (EMC) virus-induced diabetes in mice, we studied the relative importance of T cells, B cells, antibodies and macrophages in the prevention of virus-induced diabetes. Neither T cell-deficient athymic nude mice nor B cell-deficient microMT/microMT mice showed an enhanced clinical course of EMC-D virus-induced diabetes, indicating that neither T cells nor B cells played a major role in the protection against EMC-D-virus-induced diabetes. Transfer of a large amount of antiserum to EMC-D-virus-infected mice protected the development of diabetes only when transferred within 36 h of infection, the timing of which was earlier than that for the production of natural neutralizing antibodied. Since pretreatment of mice with the macrophage-activating immunopotentiator Corynebacterium parvum (CP) completely prevented the development of diabetes, we studied the clinical outcome of EMC-D-virus-infected mice pretreated with CP. Mice treated with CP showed reduced proliferation of EMC-D virus in the affected organs, including the pancreas, while the levels of development of neutralizing antibody and serum interferon were not enhanced compared with the controls. Finally, we studied the macrophages derived from mice pretreated with CP and found that they inhibited the growth of EMC-D virus in vitro more than those derived from non-treated and thioglycolate-treated mice. Taken together, it can be suggested that neither T cells nor B cells, which have to do with adaptive immunity, play a significant role in the pathogenesis of EMC-D-virus-induced diabetes, while innate immunity, which is dependent on activated macrophages, contributes to in vivo resistance against EMC-D-virus-induced diabetes.     

Successful transfer of localized autoimmunity with positively selected CD4+ cells to scid mice lacking functional B cells.

T-cell function of athymic BALB/c-nu/nu (nude) mice can be corrected by implantation of a embryonic rat thymus graft (TG) under the renal capsule (TG nude mice). However, multiple organ-localized autoimmune diseases, such as oophoritis, Sj?gren's syndrome like disease and gastritis, develop spontaneously in TG nude mice. Transfer of spleen cells from TG nude mice with such diseases leads to multiple localized autoimmune lesions with appearance of the corresponding autoantibodies in the recipient C.B-17-scid (SCID) mice. In the present study, removal of CD90+ or CD4+, but not CD8+ cells eliminated the transfer activity. Positively selected CD4+ cells proved capable of inducing lesions without the appearance of organ-specific autoantibodies, although the grade of lesions was lower than that in recipient mice that received untreated or CD8-depleted spleen cells. Target organs demonstrating CD4+ cell infiltration, generally expressed major histocompatibility complex (MHC) class II antigen (Ia) on their parenchymal cells. Injection of sera from TG nude mice with autoantibodies to SCID mice did not induce any pathogenic features in the autoantibody-target organs, although deposition of immunoglobulins in the corresponding target organs was observed. In such cases, no Ia antigens were expressed on the parenchymal cells. The data thus indicate that effector CD4+ cells can induce autoimmunity without B-cell help but that cooperation with functional B cells induces more severe tissue damage.     

The role of alpha-galactosylceramide-activated Valpha14 natural killer T cells in the regulation of Th2 cell differentiation

Valpha14 natural killer T (NKT) cells produce large amounts of both IL-4 and IFN-gamma upon stimulation with a ligand, alpha-galactosylceramide (alpha-GalCer), and play a crucial role in various immune responses, including allergic diseases. Interestingly, Valpha14 NKT cells are not essential for the induction of IgE responses but rather induce suppression of specific IgE production upon activation. The suppression in the IgE production is not detected either in Valpha14 NKT cell-deficient mice or in IFN-gamma-deficient mice. Thus, activated Valpha14 NKT cells are likely to exert a potent suppressive activity on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, little regulatory effect of IL-4 produced by Valpha14 NKT cells on Th2 cell differentiation is suggested.     

Glycolipid-activated NKT cells support the induction of persistent plasma cell responses and antibody titers

NKT cell activation with CD1d-binding glycolipid alpha-galactosylceramide (alpha-GC) enhances antibody responses to co-administered T-dependent antigen. The efficacy of alpha-GC relative to other CD1d-binding glycolipids and adjuvants is not known. There is little information on how NKT cells affect antibody production beyond initial booster-stimulated recall responses. We therefore tested the hypothesis that alpha-GC stimulates induction of plasma cells and antibody responses as effectively as Th1- and Th2-skewing variants of alpha-GC and several other adjuvants. C57BL/6 and CD1d-/- mice were immunized with nitrophenol-conjugated keyhole limpet hemocyanin (NP-KLH) plus alpha-GC or NP-KLH plus adjuvants before administration of an NP-KLH booster and assessing antibody responses and plasma cell frequency. alpha-GC boosted long-term antibody responses as efficiently as all other agents tested and induced plasma cells that were detected in bone marrow 13 weeks after immunization. We then determined whether NKT cells were required in the presence of other adjuvants. CD1d-/- mice had a reduced induction of plasma cells in response to NP-KLH/Alum as compared to C57BL/6 mice. However, NKT cells were not required for the continued presence of those cells that were induced. Although NKT cells are capable of inducing persistent plasma cell responses, they may not play a major role in supporting longevity post-induction.     

ICOS-dependent stimulation of NKT cells by marginal zone B cells

Marginal zone (MZ) B cells express high levels of CD1d molecules. In accordance, MZ B cells, like splenic conventional DCs (cDCs), efficiently trigger NKT-cell proliferation. Importantly, MZ B cells exclusively induced production of IL-4 and IL-13 by such cells whereas cDCs induced robust production of mainly IFN-γ. NKT-cell proliferation, IL-4 and IL-13 production induced by MZ B cells were dependent on ICOS/ICOS ligand interaction while IFN-γ and IL-17 induction by cDCs required glucocorticoid-induced TNF receptor/glucocorticoid-induced TNF receptor ligand interplay. Our data illustrate that both MZ B cells and cDCs act as efficient APCs for NKT cells and might differentially influence the quality of the subsequent immune response.     

Positive selection of NKT cells by CD1(+), CD11c(+) non-lymphoid cells residing in the extrathymic organs

Previously, we found that NK1.1(+), TCRalpha beta(+) natural killer T (NKT) cells develop in cytokine-supplemented suspension cultures of fetal liver established from normal, but not from beta2 microglobulin-deficient [beta2m(- / -)] mice, and that recombination-deficient SCID fetal liver can reconstiute NKT cell development in beta2m(- / -) fetal liver cultures. We found here that cells of SCID adult liver, bone marrow, spleen and thymus were able to reconstitute NKT cell development in the former culture system with efficiency comparable to normal thymic cells. The reconstitution of NKT cells was also seen in the bone marrow chimeras that had been administered a combination of beta2m(- / -) and Rag-2(- / -) bone marrow cells. Development of NKT cells was hampered by depletion of CD11c(+) or CD11b(+) cells, but not by removal of B220(+) or Gr-1(+) cells from cultures of normal fetal liver cells. Furthermore CD11c(+), CD11b(+) and / or CD11c(+) CD11b(-) cells (both populations were CD1-dull positive) enriched from Rag-2-deficient fetal livers and pulsed with alpha-galactosylceramide, a possible antigen for NKT cells, were shown to reconstitute the NKT cell development in beta2m(- / -) fetal liver cultures. Collectively, our findings suggest that non-lymphoid cells, presumably CD11c(+), CD11b(+) and / or CD11c(+), CD11b(-) dendritic cells, are involved in the mechanism of positive selection of NKT cells in the thymus and extrathymic organs.     

Rapid and preferential distribution of blood-borne alphaCD3epsilonAb to the liver is followed by local stimulation of T cells and natural killer T cells

Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body, but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of alphaCD3epsilon antibody (alphaCD3epsilonAb) we observed clear differences in antibody binding to Fcgamma receptor (FcgammaR)(+) antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne alphaCD3epsilonAb was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-beta(+) cells were positive for alphaCD3epsilonAb, and, dependent on FcgammaR-mediated cross-linking of alphaCD3epsilonAb, a similar percentage of splenic TCR-beta(+) cells were stimulated and became CD69(+). Stimulation of TCR-beta(+) cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound alphaCD3epsilonAb. In contrast to CD69 up-regulation, only CD4(+) natural killer T (NKT) cells and CD11a(high) CD8(+) T cells were activated by alphaCD3epsilonAb and expressed interferon (IFN)-gamma. Again, IFN-gamma release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells.     

Resistance of extrathymic T cells to stress and the role of endogenous glucocorticoids in stress associated immunosuppression

When mice were exposed to restraint stress for 12 or 24 h, severe lymphopenia was induced in all immune system organs, including the liver and the thymus. However, in adrenalectomized mice, this response was completely absent. Phenotypic characterization revealed that interleukin (IL)-2Rbeta+CD3int cells (i.e. extrathymic T cells) with CD4+ phenotype and the NK1.1+ subset of CD3int cells (i.e. NKT cells) in the liver as well as the mature conventional T cells in the thymus were resistant to such stress. In adrenalectomized mice, there was no significant change in the distribution of lymphocyte subsets in all tested organs before stress. Interestingly, the number of lymphocytes in the liver and spleen and the proportion of NKT cells in the liver rather increased after stress in these adrenalectomized mice. Therefore, endogenous steroid hormones were indicated to be important in the induction of immunosuppressive states after stress. Among stress associated cytokines, the secretion of tumour necrosis factor (TNF)-alpha was completely suppressed while that of IL-6 was partially suppressed in adrenalectomized mice. These results suggest that endogenous steroid hormones are important for the induction of the stress associated immunosuppression and that NKT cells are resistant to stress, namely, resistant to exposure to endogenous steroid hormones.     

Class switching and consecutive loss of dsDNA‐reactive B1a B cells from the peritoneal cavity during murine lupus development

B1a B cells have been implicated in the pathogenesis of systemic lupus erythematosus; however, their precise contribution to the disease remains unclear. Here we analysed isotype expression, organ accumulation and autoreactivity of B1a cells in the NZB/W F1 murine model for systemic lupus erythematosus using flow cytometry, ELISPOT and adoptive cell transfer. In the course of lupus, the B1a compartment is expanded and B1a cells class switch to IgG. Class‐switched B1a B cells were excluded from the peritoneal cavity but accumulated in the spleen and target organs such as kidneys and thymus. Autoreactive B1a B cells preferentially class switch, which leads to a subsequent loss of autoreactive B1a cells from the peritoneal cavity. We propose a model whereby autoreactive B1a B cells become activated early in the course of lupus and class switch to IgG. These autoreactive B1a B cells accumulate in the spleen and affected organs where they presumably secrete autoantibodies and contribute to the local and systemic pathogenesis.     

Diverse Roles of Natural Killer T Lymphocytes in Liver and Spleen in Mice with Experimental Autoimmune Uveitis

Natural killer T lymphocytes (NKT cells) have diverse roles in different organs. However, it is still unclear whether NKT cells play different roles in the liver and spleen in mice with experimental autoimmune uveitis. In the present study, we explored their diverse roles of NKT cells in the liver and spleen and found that liver-derived NKT cells could efficiently inhibit Th1 and Th17 differentiation, whereas the function of spleen-derived NKT cells was less potent. Meanwhile, the occurrence of the peak ratios of NKT cells/T cells in the spleen and liver was markedly asynchronous after immunization. Moreover, different methods of immunization could result in different immune responses in mice. Furthermore, the memory response was also found in the generation of NKT cells in mice when they received the same antigen. These results indicate that the functional roles of NKT cells possess diversity in the spleen and liver in mice with experimental autoimmune uveitis.